This application requests funds to purchase a Prairie Instruments Ultima multiphoton microscope system. This new instrument will greatly extend the current capabilities for fluorescence microscopy at Princeton University. The Prairie system will allow researchers to perform multiphoton microscopy deep in thick specimens labeled with multiple fluorescent probes at high frame rates, while reducing photodamage and avoiding loss of signal from scattering of light entering and exiting the specimen. Existing microscopes at Princeton University are conventional confocal microscopes, limiting the depth of focus and the duration of observation that is possible. The Prairie system's multiphoton frame scanning capability allows researchers to acquire dynamic colocalization data with high spatial resolution in tissues where scattering prevents one-photon methods (e.g. confocal microscopy) from penetrating. Examples include fly embryos and ovaries, developing zebrafish, and mammalian tissues ranging from bone to brain. The system accomplishes fast frame scanning by using acousto-optical devices for beam steering, thus exceeding the scan speed limit imposed by using galvanometer-mounted mirrors. Substage detection will increase the signal collection by a factor of two or more compared with light collection through the excitation objective alone. In addition to these imaging features, a second laser and set of galvanometers allow photoactivation of fluorescent proteins and photolysis of synthetic "caged" signaling molecules. Overall, these capabilities will allow Princeton researchers to track rapidly moving intracellular particles, morphological change, and biochemical dynamics on a subcellular scale with movie-rate temporal resolution. Research currently undertaken at Princeton which will benefit greatly from the Prairie system include studies of mRNA transport and cell movement in Drosophila oocytes and embryos, herpes virus assembly and transport in neuronal cells, multicellular signaling by glia and neurons in intact brain tissue, and morphogenesis of normal and cancerous ducts in breast tissue. Study of each of these biological systems will provide valuable information pertaining to significant human health problems such as birth defects, viral infection, neurological disease, and cancer.